In a typical pharmaceutical freeze-drying process, multiple vials containing a liquid drug formulation are loaded on temperature-controlled shelves within a sterile chamber and cooled to low temperatures until completely solidified. Following this freezing step, the freeze-drying chamber pressure is reduced and the shelf temperature adjusted to enable removal of the frozen solvent (i.e., drying) via sublimation in a step termed “primary drying.” When sublimation is complete, the shelf temperature is raised during “secondary drying” to remove additional un-frozen solvent bound to the solid product by e.g. adsorption. When sufficient solvent is removed, the drying process is concluded by stoppering the vials or bottles in the chamber, generally under a sub-ambient pressure of inert gas. The final dry product is called a “cake” and usually occupies the same approximate volume as the initial liquid fill due to its high porosity. The whole process usually takes multiple days to complete.
Controlling the generally random process of nucleation in the freezing stage of a lyophilization or freeze-drying process to both decrease processing time necessary to complete freeze-drying and to increase the product uniformity from vial-to-vial in the finished product would be highly desirable in the art. During the freezing step, the aqueous solution in each vial is cooled below the thermodynamic freezing temperature of the solution and remains in a sub-cooled metastable liquid state until nucleation occurs. The range of nucleation temperatures across the vials is distributed randomly between a temperature near the thermodynamic freezing temperature and some value significantly (e.g., as much as 30° C.) lower than the thermodynamic freezing temperature. This distribution of nucleation temperatures causes vial-to-vial variation in ice crystal structure and ultimately the physical, chemical, or biological properties of the lyophilized product. Furthermore, the drying stage of the freeze-drying process must be excessively long to accommodate the range of ice crystal sizes and structures produced by the natural stochastic (i.e., random or uncontrolled) nucleation phenomenon.
Additives have been used to increase the nucleation temperature of sub-cooled solutions. These additives can take many forms. It is well known that certain bacteria (e.g., Pseudomonas syringae) synthesize proteins that help nucleate ice formation in sub-cooled aqueous solutions. Either the bacteria or their isolated proteins can be added to solutions to increase the nucleation temperature. Several inorganic additives also demonstrate a nucleating effect; the most common such additive is silver iodide, AgI. In general, any additive or contaminant has the potential to serve as a nucleating agent. Lyophilization vials prepared in environments containing high particulate levels will generally nucleate and freeze at a lower degree of sub-cooling than vials prepared in low particulate environments.
All the nucleating agents described above are labeled “additives,” because they change the composition of the medium in which they nucleate a phase transition. These additives are not typically acceptable or desirable for FDA regulated and approved freeze-dried pharmaceutical products. These additives also do not provide control over the time and temperature when the vials nucleate and freeze. Rather, the additives only operate to increase the average nucleation temperature of the vials.
Equipment driven means to induce nucleation have also been attempted. Such methods have included: (i) creating ice crystals within the gas phase of the freeze-drying chamber; (ii) ultrasonic nucleation wherein mechanical vibrations or acoustic waves are imparted to the product in the vials on the freeze-dryer shelves; (iii) electro-freezing wherein an electric field is applied across electrodes submersed within the product; and (iv) vacuum induced surface freezing.
Ice crystals created within the gas phase of the freeze-drying chamber can act as nucleating agents for ice formation in sub-cooled aqueous solutions if they are transported into the liquid phase. In this “ice fog” method, a humid freeze-dryer is filled with a cold gas to produce a vapor suspension of small ice particles. The ice particles are transported into the vials and initiate nucleation when they contact the fluid interface. The “ice fog” method does not control the nucleation of multiple vials simultaneously at a controlled time and temperature. In other words, the nucleation event does not occur concurrently or instantaneously within all vials upon introduction of the cold vapor into the freeze-dryer. The ice crystals will take some time to work their way into each of the vials to initiate nucleation, and transport times are likely to be different for vials in different locations within the freeze-dryer. For large scale industrial freeze-dryers, implementation of the “ice fog” method would require system design changes as internal convection devices are required to assist a more uniform distribution of the “ice fog” throughout the freeze-dryer. When the freeze-dryer shelves are continually cooled, the time difference between when the first vial freezes and the last vial freezes will create a temperature difference between the vials, which will increase the vial-to-vial non-uniformity in freeze-dried products.
Vibration has also been used to nucleate a phase transition in a metastable material. Vibration sufficient to induce nucleation occurs at frequencies above 10 kHz and can be produced using a variety of equipment. Often vibrations in this frequency range are termed “ultrasonic,” although frequencies in the range 10 kHz to 20 kHz are typically within the audible range of humans. Ultrasonic vibration often produces cavitation, or the formation of small gas bubbles, in a sub-cooled solution. In the transient or inertial cavitation regime, the gas bubbles rapidly grow and collapse, causing very high localized pressure and temperature fluctuations. The ability of ultrasonic vibration to induce nucleation in a metastable material is often attributed to the disturbances caused by transient cavitation. The other cavitation regime, termed stable or non-inertial, is characterized by bubbles that exhibit stable volume or shape oscillations without collapse. U.S. Patent Application 20020031577 A1 discloses that ultrasonic vibration can induce nucleation even in the stable cavitation regime, but no explanation of the phenomenon is offered. GB Patent Application 2400901A also discloses that the likelihood of causing cavitation, and hence nucleation, in a solution using vibrations with frequencies above 10 kHz may be increased by reducing the ambient pressure around the solution or dissolving a volatile fluid in the solution. For large scale industrial freeze-dryers, implementation of the “ultrasonic” method poses significant system design challenges to achieve uniform distribution of the “ultrasound” energy throughout the freeze-dryer, and to maintain cleaning standards required for a cGMP sterile fill and finish manufacturing operation.
An electro-freezing method has also been used in the past to induce nucleation in sub-cooled liquids. Electro-freezing is generally accomplished by delivering relatively high electric fields (˜0.01 V/nm) in a continuous or pulsed manner between narrowly spaced electrodes immersed in a sub-cooled liquid or solution. Drawbacks associated with an electro-freezing process in typical lyophilization applications include the relative complexity and cost to implement and maintain, particularly for lyophilization applications using multiple vials or containers. Also, electro-freezing cannot be directly applied to solutions containing ionic species (e.g., NaCl).
Recently, there are studies that examine the concept of ‘vacuum-induced surface freezing’ (See e.g., U.S. Pat. No. 6,684,524). In such ‘vacuum induced surface freezing,’ vials containing an aqueous solution are loaded on a temperature controlled shelf in a freeze-dryer and held initially at about 10 degrees Celsius. The freeze-drying chamber is then evacuated to near vacuum pressure (e.g., 1 mbar) which causes surface freezing of the aqueous solutions to depths of a few millimeters. Subsequent release of vacuum and decrease of shelf temperature below the solution freezing point allows growth of ice crystals from the pre-frozen surface layer through the remainder of the solution. A major drawback for implementing this ‘vacuum induced surface freezing’ process in a typical lyophilization application is the high risk of violently boiling or out-gassing the solution under stated conditions.
Therefore, a need exists for a freeze-dryer adapted for directly controlling the nucleation of freezing in the material undergoing lyophilization. Improved control of the nucleation process can enable the freezing of all unfrozen pharmaceutical solution vials in a freeze-dryer to occur within a more narrow temperature and time range, thereby yielding a lyophilized product with greater uniformity from vial-to-vial. Controlling the lowest nucleation temperature affects the ice crystal structure formed within the vial and allows for a greatly accelerated freeze-drying process.